A multifactor complex of eukaryotic initiation factors, eIE1, eIF2, eIF3, eIF5, and initiator tRNAMet is an important translation initiation intermediate in vivo
A multifactor complex of eukaryotic initiation factors, eIE1, eIF2, eIF3, eIF5, and initiator tRNAMet is an important translation initiation intermediate in vivo
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DOI：
10.1101/gad.831800
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发表时间：
2000-10-01
影响因子：
10.5
通讯作者：
Hinnebusch, AG
中科院分区：
文献类型：
--
作者：
Asano, K;Clayton, J;Hinnebusch, AG
翻译起始因子2（eIF 2）与GTP结合，将起始剂甲硫氨酰tRNA转移到40 S核糖体亚基。eIF 5通过eIF 2/GTP/Met-tRNA（i）（Met）三元复合物在Met-tRNA（i）（Met）和起始密码子之间的碱基配对上刺激GTP水解。eIF 2、eIF 5和eIF 1都涉及严格选择AUG作为起始密码子。eIE 3与40 S核糖体结合并促进三元复合物的募集;然而，尚未观察到eIE 3和eIE 2之间的物理接触。我们表明，酵母eIE 5可以桥接eIE 3和eIF 2之间的相互作用，在体外同时结合到eIF 3亚基NIP 1的氨基末端和eIE 2 β的氨基末端的一半，依赖于一个保守的二分基序在羧基末端的eIF 5。此外，NIP 1的氨基末端可以同时结合eIF 5和eIE 1。这些发现表明存在eIE 3/eIE 1/eIE 5/eIF 2多因子复合物，该复合物在不含40 S核糖体的细胞提取物中观察到，并发现含有化学计量量的tRNA（i）（Met）。在eIE 5的二分基序中的tif 5 -7A突变破坏了多因子复合物。重要的是，tif 5 -7A突变体是温度敏感的，并显示在限制性温度下翻译起始的大幅减少。我们建议，多因子复合物是体内翻译起始的重要中间体。
Translation initiation factor 2 (eIF2) bound to GTP transfers the initiator methionyl tRNA to the 40S ribosomal subunit. The eIF5 stimulates GTP hydrolysis by the eIF2/GTP/Met-tRNA(i)(Met) ternary complex on base-pairing between Met-tRNA(i)(Met) and the start codon. The eIF2, eIF5, and eIF1 all have been implicated in stringent selection of AUG as the start codon. The eIE3 binds to the 40S ribosome and promotes recruitment of the ternary complex; however, physical contact between eIE3 and eIE2 has not been observed. We show that yeast eIE5 can bridge interaction in vitro between eIE3 and eIF2 by binding simultaneously to the amino terminus of eIF3 subunit NIP1 and the amino-terminal half of eIE2 beta, dependent on a conserved bipartite motif in the carboxyl terminus of eIF5. Additionally, the amino terminus of NIP1 can bind concurrently to eIF5 and eIE1. These findings suggest the occurrence of an eIE3/eIE1/eIE5/eIF2 multifactor complex, which was observed in cell extracts free of 40S ribosomes and found to contain stoichiometric amounts of tRNA(i)(Met). The multifactor complex was disrupted by the tif5-7A mutation in the bipartite motif of eIE5. Importantly, the tif5-7A mutant is temperature sensitive and displayed a substantial reduction in translation initiation at the restrictive temperature. We propose that the multifactor complex is an important intermediate in translation initiation in vivo.